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CNT (carbon nano tube)-loaded nucleic acid vaccine based on KHV (Koi herpesvirus) ORF149 and application

A technology of carbon nanotubes and nucleic acid vaccines, which is applied in the direction of DNA/RNA vaccination, complete cell/virus/DNA/RNA components, vaccines, etc., can solve the problems of no nucleic acid vaccine reports, etc., to improve the level of fish antibody, Immunity improvement effect

Inactive Publication Date: 2019-12-10
PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] However, in the prior art, there is no report of a nucleic acid vaccine containing ORF149 plasmid carried by carbon nanotubes against koi herpes virus KHVD

Method used

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  • CNT (carbon nano tube)-loaded nucleic acid vaccine based on KHV (Koi herpesvirus) ORF149 and application
  • CNT (carbon nano tube)-loaded nucleic acid vaccine based on KHV (Koi herpesvirus) ORF149 and application
  • CNT (carbon nano tube)-loaded nucleic acid vaccine based on KHV (Koi herpesvirus) ORF149 and application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] 1. Gene cloning

[0046] For the GenBank CyHV-3ORF149 gene sequence, use Primer Premier5.0 to design a highly specific primer pair: F: CA GAATTC CTGAGGACCATGCTCCGTC (SEQ ID NO. 1);

[0047] R: CA CTCGAG CAAGCAAGCAAAAGCAC (SEQ ID NO. 2);

[0048] The underlined parts of the pair of primers are the restriction endonucleases EcoR I and Xho I respectively, and the primers were synthesized by Sangon Biotechnology Co., Ltd.

[0049] PCR reaction system: 12.5 μL rTaq Mix, 1 μL (10 μmol / L) each of upstream and downstream primers and 2 μL DNA template, make up to 25 μL with sterilized water.

[0050] The reaction procedure is as follows: 94°C, 5min; 94°C, 30s; 55°C, 30s; 72°C, 45s; 30 cycles; 72°C, 10min. The PCR product was subjected to 1% agarose gel electrophoresis, and after gel cutting and recovery, it was ligated with the pMD18-T vector to obtain the recombinant clone plasmid pMD18-T-ORF149, and the recombinant plasmid was transformed into DH5α competent cells, and t...

Embodiment 2

[0062] Example 2 Preparation of single-walled carbon nanotube-loaded pcDNA-ORF149 nucleic acid vaccine

[0063] A carbon nanotube-loaded nucleic acid vaccine, comprising single-walled carbon nanotubes and an expression plasmid, the expression plasmid comprising pcDNA-ORF149 as an antigen; the preparation method of the pcDNA-ORF149: inserting the ORF149 gene into a gene that can be expressed in eukaryotic cells Carrier; the carrier is pcDNA-3.1(+); the mass ratio of the single-walled carbon nanotube to the expression plasmid is 8-12:1.

[0064] Preparation method of carbon nanotube-loaded nucleic acid vaccine:

[0065] (1) Mix carbon nanotubes and dimethylformamide at a mass ratio of 1:5, then add amino and paraformaldehyde at a mass ratio of 1:1, stir and mix, and place at 130°C for 96 hours to obtain a carbon nanotube mixture , and then filtered through a microporous membrane to remove unreacted carbon nanotubes and collect the filtrate. Then dissolve the residual organic s...

Embodiment 3

[0071] A method for improving fish immunity, injecting the carbon nanotube-loaded nucleic acid vaccine prepared by the present invention into the fish body, the immunity can be suppressed due to the expression of immune-related factors (cxca, illβ, mx1, vip2, IgM, Igt1) Improving by increasing; said immunity may be improved by increasing anti-koi herpes virus antibodies.

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Abstract

The invention discloses a CNT (carbon nano tube)-loaded nucleic acid vaccine based on a KHV (Koi herpesvirus) ORF149 and an application. A single-walled CNT-loaded pcDNA-ORF149 nucleic acid vaccine system is constructed successfully, fish antibody level is improved, the relative percent survival of the system is 81.9%, the immune level is far higher than that of other immune systems, and the system has the application potential in aquaculture and pathogen research.

Description

technical field [0001] The invention belongs to the field of freshwater biological vaccines, in particular to a carbon nanotube-loaded nucleic acid vaccine based on koi herpes virus ORF149 and its application. Background technique [0002] Carp herpesvirus type 3 (CyHV-3), also known as Koi herpesvirus (KHV), is a disease that causes Koi herpesvirus disease (KHVD) in carp (Cyprinus carpio Linnaeus), Koi and its variants. pathogen. The KHVD mortality rate is as high as more than 80%, which has caused huge economic losses to the carp and koi breeding industries in many countries and regions. [0003] Because there is no drug that can prevent and control koi herpes virus disease, vaccination is one of the most effective ways to control the disease. There are four types of vaccines: live, attenuated, inactivated, subunit, and DNA. [0004] Live vaccines can provide better immune protection, but may cause more virulence residues, and there are certain safety risks. Most of th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/245A61K39/385A61K47/52A61P31/22A61P37/04
CPCA61K9/0019A61K39/12A61K39/385A61K47/52A61K2039/53A61K2039/552A61K2039/60A61P31/22A61P37/04C12N2710/16034
Inventor 李莹莹王高学王庆胡峰朱斌曾伟伟王英英尹纪元
Owner PEARL RIVER FISHERY RES INST CHINESE ACAD OF FISHERY SCI
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